Plasma Mitochondrial Peptide Assays as Biomarkers of Environmental Toxin Exposure Mitochondrial dysfunction is associated with a number of chronic diseases and mitochondria are a target for numerous environmental toxins, but no validated circulating markers are available for assessing mitochondrial function or dysfunction resulting from environmental exposures. Mitochondria contain nearly a thousand proteins of nuclear origin, but the mitochondrial chromosome only encodes 13 proteins. This team recently identified a family of novel mitochondrial derived peptides (MDPs). These include humanin and six humanin-like peptides we named SHLPs, which potently regulate cell survival and metabolic processes in vitro and in vivo. It is proposed that ELISA assays for circulating humanin and other MDPs represent robust markers of mitochondrial function and will serve as early indicators of mitochondrial dysfunction associated with environmental insults. The goals in this project include continued development and characterization of ELISA assays for humanin and SHLPs in humans and mice. Preliminary data demonstrates abnormalities in their levels in human diseases states associated with mitochondrial dysfunction and in mice subjected to acute doxorubicin exposure. The in vitro effects of the mitochondrial toxins Rotenone and Doxorubicin on cell lines and primary cultures will also be examined in terms of their effect on the expression and production of mitochondrial peptides, and correlate this to measures of mitochondrial function. Finally a determination of the relationship between plasma levels of mitochondrial peptides and mitochondrial dysfunction, in mice exposed to the mitochondrial toxins Rotenone and Doxorubicin in short term and long-term studies. Changes will be assessed in circulating levels of MDPs following toxin exposure and correlated to organ-specific levels of these peptides from sacrificed animals as well as to in vivo and ex vivo analysis of mitochondrial function. Together, these studies will establish the utility of circulating mitochondrial peptide assays as markers of mitochondrial dysfunction resulting from environmental toxin exposure and will create a critical set of tools for future studies to monitor humans exposed to such toxins. This will enable prevention and intervention in subclinical stages of diseases related to mitochondrial dysfunction. PUBLIC HEALTH RELEVANCE: Relevance of this research to public health We have recently made the unique discovery that the mitochondria is the site of production of previously unrecognized peptides with biological activity that can be detected in the circulation. This represents a paradigm shift from the current approach to assessing mitochondrial function in health and disease. There is a great need to develop early biomarkers for mitochondrial dysfunction. This is true for both chronic diseases related to lifestyle and genetic susceptibility as well as to diseases related to environmental exposure. Our proposed studies will establish the utility of circulating mitochondrial peptide assays as markers of mitochondrial dysfunction resulting from environmental toxin exposure and will create a critical set of tools for future studies to monitor humans exposed to such toxins. This will enable prevention and early intervention in subclinical stages of diseases related to mitochondrial dysfunction. We believe that this project represents a highly responsive approach to the urgent concerns raised regarding the need for the development of assays for mitochondrial function that can be rapidly translated to clinical and population research.